In Brassicaceae (Cruciferae) oilseeds two classes of seed storage proteins predominate; legumin-type globulins (11S, 12S or cruciferin; Schwenke et al. 1981; Sjödahl, et al. 1991) and napin-type albumins (1.7S, 2S or napin; Lönnerdal and Janson, 1972; Crouch, et al. 1983). According to the empirical classification of Osborne (1924) based on solubility, the 11S or 12S proteins are globulins and soluble in dilute salt solutions and the 1.7S or 2S proteins are albumins and soluble in water. The 11S proteins of Brassicaceae oilseeds have Mr of 300-360 kDa and are composed of six subunits (hexameric) that are arranged as two trimers (Delseny and Raynal, 1999) which are believed to be held together by hydrogen-bonded salt bridges (Adachi et al. 2003). Each subunit of this hexameric assembly is composed of acidic or α (˜30 kDa) and basic or β (˜20 kDa) polypeptides that are linked with one disulfide bond (Delgalarrondo, 1986). The 2S proteins of Brassicaceae oilseeds has Mr of 15-18 kDa and is composed of a heavy/large (10-12 kDa) and a light/small (4-6 kDa) polypeptide that are linked by four disulfide bonds (two inter- and two intra chain) (Gehrig and Biemann, 1996; Rask et al. 1998). The 11S and 2S proteins are different in molecular structure, amino acid composition, and physico-chemical and biological properties and are thus able to provide different functionalities in practical applications.
The amount of 11S and 2S proteins of Brassicaceae species may vary. According to Crouch and Sussex (1981) cruciferin constitutes about 60% of B. napus seed protein at maturity. Recently, Malabat and group (2003) reported that among the European B. napus cultivars, the double zero (low in glucosinolates and erucic acid) varieties have higher cruciferin content (32-53% of the total proteins) than glucosinolate or erucic acid low or both contents high varieties.
Due to its high abundance and potency, Brassicaceae protein-rich meal represents a good source for recovery of plant proteins, however the allergenic potential of 2S proteins of S. alba (Sin a 1) and B. juncea (Bra j 1) is well recognized and molecular forms have been identified (Monsalve et al. 2001). The European Union has listed mustard and products as ingredients containing allergenic substances (EU Directive 2003/89/EC). The major 2S protein of B. napus, Bra n 1 shares 94% sequence similarity with Sin a 1 and Bra j 1 (Monsalve a al. 1997). Long term exposure to rapeseed meal may result in development of napin allergy in humans and napin is considered an occupational allergen (Monslave et al., 1997). Separation of 2S proteins from 11S proteins is required to produce an allergen-free protein product from Brassicaceae oilseeds. Furthermore, since 2S proteins and 11S proteins are structurally and biochemically different, once separated they can be utilized and formulated for different applications.
Known methods of Brassicaceae oilseed napin (2S) and cruciferin (11S or 12S) protein separation are primarily chromatographic separations (Schwenke et al. 1981). Recent work by Bérot et al., (2005) described a four-stage chromatographic separation and purification process that can be scaled up. This process provides high purity 11S and 2S proteins, however the process is long and complicated and has a low product yield.
Known aqueous extraction protein isolation methods for Brassicaceae seeds use solubility properties of the constituent proteins. These processes employ two aspects of protein solubility; solubility differences due to (1) pH or (2) ionic strength change. The patented methods of canola protein isolation by Newkirk et al. (2006), Diosady and group (1989; 2005) and the “Micellation process” (Murray et al. 1980; Murray, 1997; Schweizer et al., 2005; Gosnell et al. 2007) are variations of this basic behavior of proteins.
Processes patented by Newkirk et al. (2006) and Diosady and group (1989; 2005) describe solubilisation of seed proteins at pH values above neutral followed by precipitation of proteins by lowering the extract pH to acidic. In earlier studies only precipitated proteins were considered as the protein product. Later Diosady's group (Diosady et al. 1989; 2005) showed that when the majority of proteins were precipitated at acidic pH, some proteins still remained in the soluble form, which could be recovered from the remaining liquid by further processing. Newkirk et al. (2006) also describes a soluble protein fraction.
The Micellation process fractionates canola (B. napus mainly) proteins by solubilising proteins at high salt concentrations at or above neutral pH. Then a fraction of solubilised protein is recovered as hydrophobically-associated protein micelles by lowering the ionic strength in combination with bringing the pH down to mild acidic (pH 5.2-6.8) (Murray et al. 1980). Some of the proteins that remained soluble upon micelle formation can be recovered from the liquid fractions. According to Logie and Milanova (2004) the protein micelle contains primarily 7S proteins with low amounts of 12S and 2S. The liquid fraction contains primarily 2S proteins but is contaminated with 12S and 7S proteins. In legumes 7S proteins represent a distinct class of proteins which is different from 11S proteins. The legume 7S proteins have trimeric quaternary structure arrangement and no possibility of disulfide bond occurrence (Casey, 1999). In the process used by Logie and Milanova (2004) 7S protein is considered as a new protein derived from the process. It can be assumed that the conditions provided in the process may cause dissociation of hexameric 11S protein assembly into protein molecular masses that have a sedimentation coefficient of 7S, rather than true 7S proteins. However, the sedimentation coefficients of proteins are not reported for this process. The forces that keep the two trimers of the hexamer together are predominantly H-bonded salt bridges (Adachi et al. 2003) that could be disrupted due to the salting in, salting out, pH changes, temperature increase, etc. employed in this micellation process. Recent work by Gosnell et al. (2007) improved this micellation process by using a water extraction to obtain soluble proteins and then removing 7S and 12S protein by thermal treatment, pH change or ionic strength change. The remaining soluble fraction was further processed to obtain an extract consisting predominantly of 2S proteins.
The products of the above mentioned processes contain mixtures of 11S and 2S proteins. Napins and cruciferin proteins have different potential applications, due to their differences in molecular size, physico-chemical and biological properties and products containing different ratios of these two proteins may not perform these applications as well as a product containing pure or substantially pure 2S or 11S protein. Furthermore, due to the potential allergenic properties of Brassicaceae 2S protein, a method of isolating a protein extract with little or no 2S protein is required if the protein is to be used for food or other human use.